Simultaneous determination of 3′-azido-2′,3′-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography

3′-Azido-2′,3′-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3′-azido-2′,3′-dideoxyuridine (I), 5′-pivaloyloxymethyl-3′-azido-2′,3′-dideoxyuridine (II), 5′-O-valinyl-3′-azido-2′,3′-dideoxyuridine hydrochloride (III) and 5′-O-phenylalanyl-3′-azido-2′,3′-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 μM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3′-Azido-2′,3′-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 μg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25–100 μg/ml for AZDU and prodrugs II, III, and IV and 0.5–100 μg/ml for prodrug I. The limit of quantitation was 0.25 μg/ml for prodrugs II, III and IV and 0.5 μg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.